In this program, we will enable direct sequencing from RNA templates by adapting Pacific Biosciences'high throughput Single-Molecule Real-Time (SMRT) DNA sequencing to an RNA-dependent polymerase so SMRT RNA sequencing can be performed on the same instrument. SMRT sequencing provides long reads (currently 3kb and expected to increase to 10kb by the end of the program). This will have important implications to analysis of transcriptomes, crucial to all areas of medical biology. For example it will avoid cDNA conversion and its associated biases and provide the ability to examine alternative splice forms in their entirely rather than by imputed structure as with current methods. Pacific Biosciences is uniquely positioned to develop this technology because of the existing infrastructure of SMRT sequencing instrumentation and polymerase engineering resources. This program will create 3 new jobs within the next fiscal quarter, and at the point of commercialization, has the potential to create hundreds of jobs at Pacific Biosciences in delivering this technology and still more in the broader community applying it. 1 PUBLIC HEALTH RELEVANCE: The proposed research will improve researchers'ability to rapidly and cost-efficiently determine all genes that are expressed for protein synthesis in a given cell by sequencing these RNA messages. Current methods require conversion of RNA to DNA and look at fragments much smaller than the genes being studied. This research will lead to a method that allows RNA to be sequenced directly, without the degradation caused by conversion to DNA and allowing direct reading of gene-sized pieces. These two abilities will improve the quality of results;reduce the cost of studies that could be performed today, and enable studies that were previously impossible. The result will be new insights into vital processes such as development and regeneration as well as disease processes such as cancer.